High-Content Representation and Association of Three-Dimensional Cell Culture Models We will develop a platform for morphometric profiling of three-dimensional (3D) cell culture models. Multicellular systems will be imaged with confocal microscopy in full 3D; cellular organization and a number of other end points will be computed; and multidimensional phenotypic signatures will be associated with genomic data. The potential results of this initiative are (i) a basic understanding of the biological processes in a model system that is a better predictor of in vivo models, (ii) a template for drug screening against tumor lines with desirable reversion properties, and (iii) a template for hypothesis generation and validation through associations of genomic and phenotypic data. More importantly, we will design experiments that involve the alteration of mechanical properties of the microenvironment (e.g., matrix stiffness) of mammary epithelial cells. We have established that cells tune their response to matrix stiffness, proportionally increase their contractibility, promote focal adhesion assembly, and enhance growth factor signaling. The end result is that cancer-activated signaling pathways and extracellular matrix (ECM) stiffness collaborate to enhance cell tension, which compromises tissue morphology and induces malignant behavior. Therefore, identification of tension-regulated genes that are also elevated in breast tumors can serve as biomarkers for cancer diagnostic and potential therapy. Our goal is to (i) couple advanced image analysis algorithms with a bioinformatics system for high-content screening of 3D cell culture models, (ii) develop novel ways to integrate phenotypic and molecular information, and (iii) test the hypothesis that modified stromal-epithelial interactions promote tumor behavior by compromising cell and tissue phenotypes as a result of changes in the matrix stiffness. We will meet these goals in the context of a set of nonmalignant and transformed breast cell lines with significant molecular diversity and engineered matrices that induce diverse changes in cell and tissue morphology. Three-dimensional cell culture models have emerged as effective systems to study tissue differentiation and cancer behavior. If cancer is fundamentally a disease of aberrant multicellular organization, then understanding the effects of the tissue microenvironment, cellular and molecular variables, and possible therapeutic interventions on the oncogenic phenotype requires the development and use of more sophisticated models that can approximate cell-cell and cell-matrix interactions in vivo. We will develop unique technologies with important biological questions to develop the next generation of systems cell biology platforms for use with 3D cell culture assays. The deliverables of our proposed efforts are (i) a validated open source platform for routine phenotypic representation of 3D cell culture models at multiple endpoints, (ii) a seamless association of phenotypic indices with the corresponding genomic data, and (iii) an open distribution of annotated raw and processed data.